Growth hormone (GH) has long been known to be an important determinant of body growth. It has also gained much attention lately because of its potential clinical applications in reversing metabolic correlates of aging and disease. As the clinical and therapeutic potential of GH becomes apparent, it is clear that a more mechanistic appreciation of GH action is needed. The present studies will test the hypothesis that activation by GH of a GH receptor (GHR)-associated tyrosine kinase is an important component of the GHR signal transduction pathway. Two lines of evidence now strongly suggest that GHR forms a dissociable complex with a tyrosine kinase rather than having intrinsic tyrosine kinase activity. First, GH-GHR complexes from cells expressing cloned liver GHR (which does not have a consensus sequence for an ATP binding site) co-purify with and are phosphorylated by a tyrosine kinase. The amount of tyrosine kinase available appears to be cell-type specific. Second, tyrosine kinase activity can be dissociated from, and then reconstituted with, GHR. These preliminary results shall be verified and the GHR-kinase identified by size. The important mechanistic question of whether GH binding to GHR increases the association of GHR with GHR-kinase and/or increases the activity of the GHR-kinase will be addressed. Using antibodies to known tyrosine kinases, it will be determined whether the GHR is a known kinase (e.g. Src family). If it cannot be established that the GHR-kinase is a known kinase, experiments will be initiated to clone the GHR kinase. Expression libraries will be screened for proteins that bind to GHR cytoplasmic domain, or portions thereof. If larger amounts of the GHR-kinase are required for identification or cloning, GHR affinity columns will be used to affinity purify GHR-kinase. GHR will be obtained by overexpressing cloned liver GHR in a baculovirus system, or the cytoplasmic domain of GHR in E. coli if the GHR-cytoplasmic domain is shown to associate with GHR-kinase when expressed as a dominant negative mutant. The importance of tyrosine kinase activity in actions of GH will be examined by testing whether inhibitors of the GHR-kinase cause loss of specific functions of GH. The effect of mutating specific GHR tyrosyl residues on specific functions of GH will be tested to evaluate the importance of GHR tyrosyl phosphorylation itself. Substrates of the GHR-kinase, and proteins whose association with GHR may be affected by phosphorylation of GHR tyrosyl residues, will also be identified and characterized. These studies will provide greatly needed insight into the complex mechanism by which GH brings about its diverse effects.